With a long-term goal to better understand the molecular mechanisms of epithelial innate immune responses to pathogens, we will investigate innate immunity in cholangiocytes (epithelial cells lining the biliary tract) in response to Cryptosporidium parvum, an NIAID Category B Priority Pathogen that causes both intestinal and biliary disease in humans. Using an in vitro model of human biliary cryptosporidiosis established by us, we have demonstrated that: (i) two Toll-like receptors (TLRs), TLR2 and TLR4, and an associated intracellular signaling pathway (NF-kappaB activation) play a central role in C. parvum recognition and induction of innate immune responses (e.g., release of cytokines/ chemokines) in cholangiocytes;(ii) C. parvum infection alters cholangiocyte expression of specific endogenous microRNAs (miRNAs), a newly identified class of small regulatory RNAs important in post-transcriptional gene regulation;and (iii) TLR/NF-kappaB signals are involved in C. parvum-induced cholangiocyte miRNA expression. Thus, we will test the CENTRAL HYPOTHESIS that epithelial innate immunity in response to C. parvum infection involves TLR-mediated pathogen recognition and subsequent alteration of miRNA-mediated post-transcriptional gene regulation. In our three integrated SPECIFIC AIMS, we will test the hypotheses that: (i) C. parvum activation of host-cell TLRs alters cholangiocyte expression of endogenous miRNAs;(ii) C. parvum-induced miRNA expression in cholangiocytes is mediated by TLR activation of the nuclear transcription factor, NF-kappaB;and (iii) C. parvum-induced alterations in miRNA expression influence associated post-transcriptional gene regulation and contribute to cholangiocyte innate immune responses. Innovative aspects of the application include novel methodologies for miRNA analysis, an in vitro model of human biliary cryptosporidiosis, and new concepts (regulation of miRNA expression by transcription factors and miRNA-mediated post-transcriptional gene regulation in epithelial innate immunity). These studies will address a fundamental question related to epithelial innate immunity to a Category B Pathogen;specifically, what is the role of miRNAs in TLRmediated cholangiocyte innate immune responses to C. parvum? This NEW CONCEPT is likely relevant to innate and adaptive immunity in general and our results could also provide a rational basis for the design and implementation of new therapeutic strategies for microbial infection.